THU0127 Functional activity of membrane-bound glucocorticoid receptors. (23rd January 2014)

• Record Type: Journal Article
• Authors: Strehl, C.; Gaber, T.; Jakstadt, M.; Hahne, M.; Hoff, P.; Burmester, G.-R.; Buttgereit, F.
• Is Part Of: Annals of the rheumatic diseases. Volume 71(2012)Supplement 3
• Page Start: 198
• Page End: 198
• Publication Date: 2014-01-23
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2012-eular.2092
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: Glucocorticoids are among the most commonly used anti-inflammatory and immunosuppressive drugs in the treatment of rheumatic diseases. They exert their anti-inflammatory and immunosuppressive effects primarily via the cytosolic glucocorticoid receptor (cGR), but also via rapid, specific and unspecific non-genomic actions. The latter actions are hypothesized to be in part mediated by membrane-bound glucocorticoid receptors (mGR), which are up-regulated in monocytes from patients with inflammatory diseases such as RA, SLE and AS. We have previously shown that the human GR gene encodes the expression of both the cGR and the mGR protein. We also showed that the activation of the mGR resulted in rapid signalling via (de)phosphorylation events. Objectives: Here, we investigated more in detail the mechanisms of this rapid mGR signalling, and the subsequent effects on the genome. Methods: Human monocytes (obtained from healthy donors) were stimulated with LPS (2μg/ml) for 24 hours in order to increase mGR expression up to 70%. Subsequently, cells were treated with membrane impermeable dexamethasone bound to BSA (10 -9 M) (DEX-BSA), DEX (10 -8 M) or BSA (10 -9 M) alone. The incubation time was 20 minutes for kinome analysis and 3 h 20 min for genome analysis. Using the PepChip™ array technique, we identified kinases solely triggered by DEX-BSA. These kinases were classified according to their molecular and biological functions via the Panther database. In order to analyse mGR-mediated effects on gene expression, we analysed lysates of the cells treated with DEX-BSA, DEX or BSA alone by DNA microarray . The results obtained were validated by RT-qPCR. Functional analysis of the data was performed using the Panther database. Results: The analysis of the kinome of LPS stimulated human monocytes demonstrated DEX-BSA to induce rapid (de)phosphorylation events of proteins belonging to signalling cascades. We identified a number of MAP-kinases, including p38-MAPK, protein kinase A and C as well as casein-kinase 2 which have to be considered as possible upstream kinases being responsible for the modification of peptide-substrates. Increased phosphorylation of p38-MAPK due to DEX-BSA treatment was observed in phosphoprotein detection Bio-Plex assay as well as in Western blot analysis. Focusing on the functional activity of the mGR, genome analysis of LPS stimulated human monocytes revealed that DEX-BSA also alters gene expression. Validation of these findings via RT-qPCR confirmed that predominantly cell surface receptor linked signal transduction pathways, intracellular signalling cascades and processes involved in the metabolism of proteins, nucleosides, nucleotides and nucleic acids are affected. Conclusions: The human mGR is functionally active as shown by rapid effects on the kinome as well as on the genome. These effects need to be further investigated in order to identify mGR signalling pathways and their targeted genes more in detail. Disclosure of Interest: None Declared